神经干细胞在wistar大鼠体内、外追踪胶质瘤细胞的特性

目的 观察在胶质瘤细胞存在这一病理状态下,神经干细胞(neural stem cells,NSCs)在体内、外的迁移特点;了解导入NSCs的外源基因在迁移过程中的表达情况。方法 分离、培养NSCs,脂质体介导pIRES2-EGFP质粒转染NSCs,BrdU标记NSCs;共同培养胶质瘤细胞和NSCs,观察NSCs在体外的迁移特点;制作大鼠颅内胶质瘤模型,用转染EGFP基因和BrdU标记的NSCs接种颅内胶质瘤,免疫组织化学检测,观察NSCs在体内的迁移特点,了解导入NSCs的外源基因在颅内的表达情况。结果 培养成功了Wistar胎鼠大脑NSCs,成功将EGFP基因导入NSCs,NSCs在体内、外显示了对胶质瘤细胞强烈的追踪特性,同时表达外源基因的产物。结论 NSCs在体内、外具有追踪胶质瘤细胞的特性,同时能够表达外源基因的产物。     

白细胞介素24对大鼠脑胶质瘤细胞Survivin表达的影响

目的探讨白细胞介素24(IL-24)基因对荷瘤大鼠脑胶质瘤细胞Survivin表达的影响。方法 48只SD大鼠随机分为实验组和对照组,每组24只,分别接种IL-24/C6细胞(转染IL-24基因的C6鼠脑胶质瘤细胞)和C6鼠脑胶质瘤细胞。接种21 d后,采用RT-PCR、Western blot和免疫组化方法检测两组大鼠脑肿瘤组织中Survivin mRNA和蛋白的表达,采用流式细胞学检测肿瘤细胞凋亡率。结果 RT-PCR检测显示:实验组大鼠脑肿瘤组织中Survivin mRNA表达水平明显低于对照组(P<0.01)。Western blot及免疫组化法检测显示:实验组大鼠脑肿瘤组织中Survivin的蛋白水平明显低于对照组(P<0.01)。流式细胞学结果显示:实验组细胞凋亡率显著高于对照组(P<0.01)。结论 IL-24可抑制大鼠胶质瘤中Survivin mRNA和蛋白的表达,促进细胞凋亡,抑制肿瘤生长。     

绿色荧光蛋白报道基因在鼠C6胶质瘤细胞中的应用研究

目的 :探讨绿色荧光蛋白 (GFP)报道基因转染C6鼠胶质瘤细胞的体内外表达及对细胞生物学性状的影响。方法 :荧光相差显微镜筛选稳定表达GFP的克隆 ,流式细胞术分析细胞周期。将已转染和未转染瘤细胞植入SD大鼠脑内 ,建立动物模型 ,定期随机处死大鼠 ,鼠脑标本行病理学、增殖与凋亡的检测 ,激光共聚焦显微镜检查肿瘤细胞内荧光强度及其分布。结果 :GFP在C6瘤细胞的体外和体内均获得长期稳定表达 ,检测肿瘤细胞的敏感性及特异性优于HE染色。结论 :GFP对肿瘤细胞生物学性状无影响是比较理想的报道基因     

转染外源性p16基因对恶性胶质瘤生长抑制作用的动物体内实验研究

目的 :探讨p16基因在体内对恶性胶质瘤的生长抑制作用。方法 :将外源性p16基因导入C6 细胞内 ,应用立体定向技术将C6细胞种植于SD大鼠尾状核头部 ,用核磁共振 (MR)扫描技术 ,动态观察颅内肿瘤生长情况。并通过免疫组化、原位杂交和细胞凋亡检测肿瘤细胞的增殖活性。结果 :转染组和治疗组大鼠生存期较对照组明显延长。治疗组肿瘤随时间的延长逐渐缩小。免疫组化显示转染组和治疗组P16蛋白表达明显增强。原位杂交和细胞凋亡检测表明 ,转染组和治疗组大鼠肿瘤细胞增殖活性降低。结论 :p16基因在体内有抑制恶性胶质瘤生长的作用 ;瘤体内注入p16cDNA质粒 脂质体复合物 ,可使肿瘤生长受到明显抑制 ,并使多数肿瘤消失     

大鼠C6脑胶质瘤模型的病理特征与MRI的观察

目的建立SD大鼠C6胶质瘤模型并对其病理特征及MRI进行观察。方法50只SD大鼠随机分成5组，每组10只，c6胶质瘤细胞悬液立体定向接种于大鼠的右侧尾状核，接种后观察大鼠的生活状态、生存期；分别于接种后不同时段进行MRI观察肿瘤生长特性及肿瘤体积的测量；取不同时段组大鼠脑标本行脑组织HE染色、透射电镜(transmission electron microscope，TEM)、脑组织含水量测量(与10只正常大鼠对照)、胶质纤维酸性蛋白(GFAP)免疫组化检查。结果立体定向颅内接种成功率97．5％，未见远处及颅外转移，肿瘤在一定时期内牛长较快，脑水肿随肿瘤的生长明显加重，生存期观察组中7只荷瘤鼠死亡，3只肿瘤自发部分消退。结论立体定向建立大鼠C6胶质瘤模型成功率高，接种后颅内肿瘤呈浸润性生长，与人脑胶质瘤具有相似性，由于生存期观察组中有部分荷瘤鼠出现肿瘤自发部分消退，故应用该模型评价治疗效果时应慎重；TIWI增强扫描可清晰显示肿瘤影像，且能更早发现肿瘤；MRI联合病理可较好反映肿瘤生长方式及发展过程。     

PTEN-反义AKT2联合基因治疗胶质瘤的体内外实验研究

目的研究PTEN-反义AKT2的联合基因治疗在动物体内、体外抑制脑胶质瘤生长的效果。方法将大鼠C6胶质瘤细胞进行PTEN-反义AKT2的共转染,进行了原位杂交等体外实验研究,并进行了大鼠的体内实验研究。结果PTEN与反义AKT2共转染在体外可以产生比较明显的协同作用,在体内实验中,对照组和空载组大鼠均于3周内死亡;转染组8只大鼠和治疗组7只大鼠观察60d内无自然死亡。结论PTEN-反义AKT2联合基因治疗可以产生明显的协同作用,与单一治疗效果相比抑瘤作用明显增强,可以作为联合基因治疗的候选基因组合。     

Apoptotic cell death induced by local brain hyperthermia in a rat glioma model

Hyperthermia has been shown to inhibit glioma growth both in vitro and in vivo, and has been reported to induce apoptosis of a variety of cells. We investigated the role of apoptosis in tumor cell death following hyperthermia in a rat glioma model representing human glioblastoma. Apoptotic cell death was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and hematoxylin and eosin (H & E) staining. We also examined c-Jun expression immunohistochemically. Apoptotic cell death in rat brain tumors that grew after implantation of C6 glioma cells showed regional differences. In all rats, apoptotic cells, characterized by extreme chromatin condensation and fragmented nuclei with apoptotic bodies in H & E-stained sections, were observed in the gliomas’ necrotic cores. TUNEL-positive cells were observed in the border zones between necrotic and vital tumor cells. Before hyperthermia, TUNEL-positive cells were sporadically distributed in the vital tumor tissue. After hyperthermia, the number of TUNEL-positive cells in the peripheral region of the tumor mass increased significantly, reached a peak after 6 h and returned to the basal level within 24 h (P < 0.01). C-Jun protein immunoreactivity was not observed in the cells at the tumor periphery. These data indicate that significantly apoptotic cell death unrelated to c-Jun expression occurs after hyperthermia, and that this form of cell death may be the mechanism of tumor regression following hyperthermia treatment of intracranial gliomas. Received: 8 December 1997 / Revised, accepted: 27 March 1998     

大鼠脑干胶质瘤模型的建立及MRI特点

目的 建立大鼠脑干胶质瘤模型,并研究成瘤鼠运动功能及胶质瘤MRI成像特点.方法 采用大鼠脑立体定向仪,将C6胶质瘤细胞接种至SD大鼠脑干内.MRI动态观察肿瘤生长情况,并观察接种C6胶质瘤细胞后大鼠的运动能力、生存周期等.结果 C6胶质瘤细胞接种成功率为100%.生存分析表明:大鼠于接种后第16～22天死亡.接种后第7天,MRI即可检出肿瘤生长,荷瘤鼠的运动能力随着成瘤时间的推移逐渐减弱.结论 大鼠脑干胶质瘤模型成瘤率高,有良好的可重复性和可预测性.MRI观察该模型具有脑干胶质瘤成像的特点,细胞接种后10～18 d是最佳观测期.     

反义AKT2 cDNA治疗C6胶质瘤的体内外实验研究

目的研究反义AKT2(antisense AKT2，AS—AKT2)cDNA对鼠脑胶质瘤细胞系C6的生长抑制作用。方法将AS—AKT2 cDNA构建体转染鼠脑胶质瘤细胞系C6，原位杂交和蛋白印迹鉴定后，应用PCNA阳性率和MTT法检测细胞增殖能力，TUNEL法计算凋亡指数。应用立体定向技术将C6细胞和转染反义AKT2 cDNA的C6细胞种植到SD大鼠的右侧尾状核作为对照组和转染组；并对颅内已经形成C6胶质瘤的大鼠进行脂质体包裹的AS—AKT2 cDNA和空载体治疗；MRI动态监测大鼠颅内肿瘤生长情况，并检测标本AKT2和PCNA表达以及细胞的凋亡情况。结果转染AS—AKT2 cDNA后C6细胞AKT2表达显著抑制，增殖减慢，凋亡指数增加。反义治疗组和转染组大鼠生存时间明显延长；转染组和治疗组肿瘤标本AKT2表达下降或消失，PCNA阳性率降低，可见大量凋亡细胞，而对照组和空载组标本几乎没有凋亡细胞。结论体内外实验证明AS—AKT2 cDNA可以抑制肿瘤细胞增殖、诱导凋亡，AKT2可作为基因治疗胶质瘤的重要优选靶的。     

真核表达载体pIRES2-EGFP-p16的构建及其在胶质瘤细胞C6内的表达

目的 构建真核表达载体pIRES2-EGFP-p16,并在神经胶质瘤细胞系C6中进行表达.方法 用PCR的方法扩增p16基因,构建真核表达载体pIRES2-EGFP-p16,经BamH I及Xho I双酶切鉴定并测序.通过脂质体法转染C6细胞,用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用免疫组化染色的方法检测细胞中p16的表达.结果 成功地构建了真核表达载体pIRES2-EGFP-p16,用脂质体法转染神经胶质瘤细胞C6后,经荧光显微镜和免疫组化染色法检测,可见细胞内有EGFP及p16的表达.结论 成功地构建真核表达载体pIRES2-EGFP-p16,并在神经胶质瘤细胞C6中表达,为研究p16对肿瘤的生物学作用以及p16在肿瘤基因治疗中的应用奠定了基础.

Abstract：

Objective To construct the eukaryotic expression vector pIRES2-EGFP-p16,and to express p16 in glioma cell C6. Methods The p16 gene was amplified by PCR using pCMV5-HA-p16 as a template,and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-p16 was constructed by introducing p16 DNA fragment into the sites of BamH I and Xho I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the P16 protein expression was detected by immunostaining using anti-P16 antibody. Results The eukaryotic expression vector pIRES2-EGFP-p16 was constructed and transfected successfully into C6 glioma cells. The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and P16 protein was found in the plasma and nuclear. Conclusion The recombinant expression vector pIRES2-EGFP-p16 was constructed, and the EGFP and p16 gene could be co-expressed in the C6 cells. This study laid a foundation for the further research of the function of p16 in cell differentiation, growth and tumorigenesis.     

Annexin A2 promotes glioma cell invasion and tumor progression

Gliomas are highly invasive, lethal brain tumors. Tumor-associated proteases play an important role in glioma progression. Annexin A2 is overexpressed in many cancers and correlates with increased plasmin activity on the tumor cell surface, which mediates degradation of extracellular matrix and promotes neoangiogenesis to facilitate tumor growth. In this study, we used two glioma cell lines, mouse GL261-EGFP and rat C6/LacZ, as well as stable clones transfected with an annexin A2 knockdown construct. We find that the annexin A2 knockdown decreased glioma cell migration in vitro and decreased membrane-bound plasmin activity. In vivo, we injected the glioma cells into the rodent brain and followed glioma progression. Knockdown of annexin A2 in glioma cells decreased tumor size and slowed tumor progression, as evidenced by decreased invasion, angiogenesis, and proliferation, as well as increased apoptosis in the tumor tissue of the annexin A2 knockdown group. Moreover, we report that the levels of expression of annexin A2 in human glioma samples correlate with their degree of malignancy. Together, our findings demonstrate that inhibition of annexin A2 expression in glioma cells could become a new target for glioma therapy.     

大鼠树突状细胞疫苗治疗脑胶质瘤的实验研究

目的研究树突状细胞疫苗对C6荷瘤大鼠的免疫治疗效应,并比较免疫治疗策略的异同。方法将成年健康SD大鼠40只平均分为4组,分别经RN A致敏和C6冻融抗原致敏的DC免疫以及对照组经未致敏D C和R PM I1640免疫,采用LD H试剂盒检测CTL的体外杀伤活性。建立大鼠C6脑胶质瘤颅内肿瘤模型,观察其生存期,并进行病理学检查。结果肿瘤RN A致敏的D C疫苗活性最强,与C6冻融抗原致敏的DC疫苗相比,CTL杀伤活性差异不显著(P>0.05),但与对照组相比,差异非常显著(P<0.01)。治疗组荷瘤大鼠生存时间与对照组相比,差异非常显著(P<0.01);两治疗组之间差异不显著(P>0.05)。治疗组肿瘤生长明显抑制。结论C6冻融抗原和肿瘤细胞R NA致敏的树突状细胞疫苗均是有效的免疫治疗方法。     

立体定向技术建立大鼠脑胶质瘤激光间质热疗模型

目的 利用立体定向技术接种SD大鼠C6脑胶质瘤,并建立脑胶质瘤激光间质热疗(LITT)模型.方法 采用立体定向技术,将体外培养并调制的C6胶质瘤细胞悬液20μl(浓度1×10~(11)/L)接种于SD大鼠右侧尾状核区.分时段MRI检查;做组织病理学和Ⅷ因子相关抗原(FⅧR),胶质纤维酸性蛋白(GFAP)和S-100蛋白免疫组化检查.根据MRI扫描监测,校正肿瘤定位,按2～10 W不同功率和热疗时间分组,插入半导体激光光纤进行间质热疗,同时使用ThermaCAM S65型红外热像仪测量肿瘤的中心靶点皮层温度和(或)热电偶仪间质测量靶区周边的深部温度.结果 优化的立体定向接种技术使本组大鼠脑胶质瘤动物模型具有颅内生长稳定,成瘤率高,未见颅外转移病灶,实验周期短,可重复性好,可插入热疗光纤热疗,组织学上接近人类特征.LITT各组靶区温度高于假手术组(P<0.05);在同一治疗组内中心靶点皮层温度和靶区周边的深部温度之间有近似性,差异无统计学意义(P>0.05).结论 利用立体定向技术可成功建立SD大鼠脑尾状核C6胶质瘤模型,其肿瘤MRI影像及病理特征与人脑胶质瘤相似.红外热像测温技术在大鼠实验性LITT研究中的应用具有可行性,可联合热电偶深部测温技术应用于脑肿瘤LITT治疗.     

9L/Wistar大鼠脑胶质肉瘤模型的建立

目的建立稳定的9L／Wistar大鼠脑胶质肉瘤模型。方法构建稳定表达萤火虫荧光素酶PGL的9L细胞株9L^lvc，采用立体定向手术，在Wistar大鼠右侧尾状核接种DAPI荧光标记的9L^lvc细胞，分别于接种后第7，14，21天通过Xenogen活体动物体内成像系统检测肿瘤的生长情况，并断头取材，观察肿瘤的形态学变化，采用免疫组化的方法检测肿瘤细胞胶质纤维酸性蛋白（GFAP）、波形蛋白（Vemintin）的表达。结果大鼠接种9L^lvc细胞后成瘤率100％。活体成像显示细胞接种后第7天到第21天肿瘤呈逐渐增大趋势。组织切片结果显示肿瘤与周围脑组织边界较清楚，未见包膜；瘤内新生血管丰富，可见出血。肿瘤组织免疫组织化学GFAP及Vemintin蛋白染色阴性。结论该方法建立的大鼠脑胶质肉瘤模型稳定可靠，符合恶性胶质肉瘤的生物学特征．是理想的脑胶质肉瘤实验研究材料。     

利用C6胶质瘤干细胞立体定向建立大鼠脑胶质瘤模型

目的利用C6胶质瘤干细胞建立wistar大鼠脑胶质瘤模型,观察肿瘤生长规律及病理特征,探讨利用肿瘤干细胞构建模型的优势。方法体外提取并培养c6细胞系中胶质瘤干细胞,应用立体定向法在鼠脑右侧尾状核区接种10^4个胶质瘤干细胞,术后连续观察大鼠生存状态和生存时间,分时段行鼠脑MRI检查、病理HE切片及GFAP免疫组织化学检查,观察肿瘤生长及病理特征。结果利用本方法建立模型成瘤率为100％,术后大鼠恢复快,术后反应轻,未见颅外转移,生存期稳定,重复性好,肿瘤生长规律、影像学特征及组织病理特点与人脑胶质瘤相似。结论利用胶质瘤干细胞建立的大鼠脑胶质瘤模型稳定可靠,肿瘤生长更贴近胶质瘤在脑内自然发生的特点,且较传统方法脑内注射体积小,模型制作时间短,术后恢复快,成瘤率更高等优点,为研究胶质瘤干细胞在肿瘤形成过程中的生长方式、分子机制及实验性治疗提供了一个较为理想的模型。     

Prolonged survival of rats with intracranial C6 gliomas by treatment wit~ TGF-β antisense gene

Abstract

Using an intracranial rat C6 glioma model, we tested the hypothesis that gene modification ofglioma cells to block the expression of the immunosuppressive cytokine TGF-[3 (transforming growth factor (3) may enhance anti-tumor immune responses and thereby prolong survival of tumor-bearing animals. The cDNA for simian TGF-β2 was ligated in antisense orientation into the episomal plasmid mammalian expression vector pCEP-4. This TGF-β-antisense vector was transfected into C6 glioma cells by standard electroporation techniques. PCR was used to determine that the rat C6 clones were successfully transfected with the antisense-TGFβ construct. Twenty-nine adult female Wistar rats harboring 7-day-old intracranial C6 tumors were then subcutaneously injected with either saline (n = 9), unmodified C6 glioma cells (n =10), or TGF-β-antisense-modified C6 cells (n =70). Animals were followed, for survival, and Fisher!s exact method was used to interpret the significance of differences between experimental groups. The survival of tumor-bearing rats injected with TGF-β-antisense-modified C6 cells was significantly prolonged! relative to the survival of rats receiving injections of saline or unmodified C6 cells alone. Six of the ten (60%) TGF-β-antisense treated animals survived for 72 weeks! whereas none of the nine (0%) animals treated with saline and none of ten (0%) of those treated with C6 cells alone survived past 5 weeks. These results indicate that the genetic inhibition of immunosuppressive cytokines (such as TGF-(3) may reverse the phenotypic immunosuppression caused by such factors, and thereby prolong the survival of C6 tumorbearing animals. Future investigations using cytokine gene modifications in other brain tumor models are warranted. [Neural Res 1998; 20: 742–747]     

miR-103a-3p过表达靶向负调控PDK4抑制胶质瘤生长及侵袭

目的 探讨miR-103a-3p过表达对胶质瘤C6细胞恶性生物学行为的影响及对裸鼠移植瘤生长的影响。方法 体外培养鼠源性C6胶质瘤细胞,转染miR-103a-3p mimics质粒过表达miR-103a-3p,转染miR-103a-3p mimics+pcDNA-PDK4质粒分析PDK4过表达对miR-103a-3p过表达的影响;EDU法检测细胞增殖活性;qRT-PCR检测miR-103a-3p、PDK4 mRNA表达水平;流式细胞仪检测细胞凋亡率;Transwell实验检测细胞侵袭能力;免疫印迹法检测E-cadherin、N-cadherin、vimentin蛋白表达水平。取40只裸鼠,其中20只皮下注射未转染质粒的C6细胞、20只皮下注射转染miR-103a-3p mimics质粒的C6细胞构建移植瘤模型,分析miR-103a-3p mimics过表达对移植瘤生长的影响。结果 生物信息学及双荧光素酶试验证实PDK4是miR-103a-3p的靶点。过表达miR-103a-3p明显降低C6细胞PDK4、Ki67、PCNA、N-cadherin、vimentin表达水平（P<0.05）,明显抑制C6细胞增殖活性、侵袭能力（P<0.05）,明显增加C6细胞E-cadherin表达水平、细胞凋亡率（P<0.05）。过表达PDK4明显抑制过表达miR-103a-3p对C6胶质瘤细胞的作用（P<0.05）。过表达miR-103a-3p明显抑制裸鼠移植瘤生长（P<0.05）,抑制肿瘤组织PDK4、Ki67、vimentin表达（P<0.05）。结论 过表达miR-103a-3p通过靶向抑制PDK4表达,一方面抑制胶质瘤细胞增殖、促进胶质瘤细胞凋亡,从而抑制胶质瘤生长;另一方抑制胶质瘤上皮-间质转化过程,从而抑制胶质瘤细胞侵袭。     

肿瘤干细胞致敏的树突状细胞对脑胶质瘤细胞免疫的影响

目的 研究肿瘤干细胞(BTSCs)致敏的树突状细胞(DCs)疫苗对颅内荷瘤小鼠的治疗作用.方法 无血清培养基中加入表皮生长因子和碱性成纤维细胞生长因子,将C6胶质瘤细胞诱导成胶质瘤干细胞,以此致敏大鼠骨髓来源的树突状细胞制备疫苗;立体定向建立大鼠颅内C6胶质瘤模型,分为A、B、C、D四组.每组分别经尾静脉注射1×107BTSCs致敏的DCs(DCs-BTSCs)、1×107 C6胶质瘤细胞致敏的DCs(DCs-C6)、1×107DCs及PBS,Kaplan-Meier法对大鼠生存情况进行分析,大鼠脑组织标本行HE染色及免疫组化分析.结果 胶质瘤干细胞CD133+及nestin染色阳性;DCs具有典型的树突状结构,特异性标志OX62+表达阳性;生存时间A组较其他组明显延长,Log-rank检验差异有统计学意义(P＜0.05);A组HE染色炎性细胞浸润最多,免疫组化可见较多的CD8+T淋巴细胞.结论 肿瘤干细胞致敏的树突状细胞疫苗能明显提高机体对胶质瘤的免疫力,其作用优于胶质瘤细胞致敏的树突状细胞疫苗,为树突状细胞疫苗的临床应用提供了新的依据.

Abstract：

Objective To investigate the effect of dendritic cells pulsed with brain tumor stem cells which are used to treat on intracranial glioma.MethodWe obtained murine brain tumor stem cells by growing C6 cells in epidermal growth factor/basic fibroblast growth factor without serum.Dendritic cells isolated from rat bone marrow were pulsed with BTSCs.Rat brain glioma models were established by stereotactic technique.107 DCs pulsed with BTSCs and C6 were injected through tail vein in group A and group B respectively.The same number of DCs and the same volume PBS were applied to group C and group D.The survival time of rats was analyzed by Log- rank survival analysis.Tumor samples were examinedwithHE staining and immunohistochemistry.Methods BTSCs expressedCD133 + and nestin.DCs appeared typical long dentrite in morphology and expressed OX62+ markers.The survival analysis showed group A was statistically significant in constrast to the other goups (P<0.05).The tumors in group A have the most inflammation and CD8 + T lymphocytes.Concluion DCs loading with BTSCs lysates can provide a higher level of immunity protect against gliomas than those loading with C6 cells,which provide a new method in the immuntherapy of brain glioma.